Summary of Study ST000393

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000307. The data can be accessed directly via it's Project DOI: 10.21228/M8Z01Z This work is supported by NIH grant, U2C- DK119886.

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Study IDST000393
Study TitleLong-Chain Fatty Acid Combustion Rate Is Associated with Unique Metabolite Profiles in Skeletal Muscle Mitochondria
Study SummaryIncomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA β-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate. This proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle.
Institute
University of California, Davis
DepartmentGenome and Biomedical Sciences Facility
LaboratoryWCMC Metabolomics Core
Last NameFiehn
First NameOliver
Address1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Emailofiehn@ucdavis.edu
Phone(530) 754-8258
Submit Date2016-05-06
Publicationshttp://dx.doi.org/10.1371/journal.pone.0009834
Raw Data AvailableYes
Raw Data File Type(s)peg
Analysis Type DetailGC-MS
Release Date2016-06-18
Release Version1
Oliver Fiehn Oliver Fiehn
https://dx.doi.org/10.21228/M8Z01Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR000307
Project DOI:doi: 10.21228/M8Z01Z
Project Title:Long-Chain Fatty Acid Combustion Rate Is Associated with Unique Metabolite Profiles in Skeletal Muscle Mitochondria
Project Summary:Incomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA β-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate. This proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle.
Institute:University of California, Davis
Department:Genome and Biomedical Sciences Facility
Laboratory:WCMC Metabolomics Core
Last Name:Fiehn
First Name:Oliver
Address:1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Email:ofiehn@ucdavis.edu
Phone:(530) 754-8258
Funding Source:NIH U24DK097154
Publications:http://dx.doi.org/10.1371/journal.pone.0009834

Subject:

Subject ID:SU000414
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57Bl/6J
Age Or Age Range:4 months
Gender:Female
Human Smoking Status:Former/Current
Animal Animal Supplier:Jackson Laboratories
Animal Housing:housed in a facility at 23°C
Animal Light Cycle:12∶12 hr light cycle, lights on 0700
Animal Feed:with free access to chow (4.5% fat/weight), for at least 1 week before being studied
Species Group:Mammal

Factors:

Subject type: Animal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Organ Time Dose
SA018676061215byusa1091_1cytosol 0 min palmitic acid 2.4 uM
SA018677061215byusa1127_1cytosol 0 min palmitic acid 2.4 uM
SA018678061215byusa1092_1cytosol 0 min palmitic acid 2.4 uM
SA018679061215byusa1119_1cytosol 0 min palmitic acid 2.4 uM
SA018680061215byusa1096_1cytosol 0 min palmitic acid 2.4 uM
SA018681061215byusa1088_1cytosol 0 min palmitic acid 2.4 uM
SA018682061215byusa1134_1cytosol 0 min palmitic acid 9.6 uM
SA018683061215byusa1090_1cytosol 0 min palmitic acid 9.6 uM
SA018684061215byusa1086_1cytosol 0 min palmitic acid 9.6 uM
SA018685061215byusa1103_1cytosol 0 min palmitic acid 9.6 uM
SA018686061215byusa1079_1cytosol 0 min palmitic acid 9.6 uM
SA018687061215byusa1065_1cytosol 0 min palmitic acid 9.6 uM
SA018688061215byusa1112_1cytosol 0 min palmitoyl carnitine 9.6 uM
SA018689061215byusa1075_1cytosol 0 min palmitoyl carnitine 9.6 uM
SA018690061215byusa1124_1cytosol 0 min palmitoyl carnitine 9.6 uM
SA018691061215byusa1087_1cytosol 0 min palmitoyl carnitine 9.6 uM
SA018692061215byusa1077_1cytosol 0 min palmitoyl carnitine 9.6 uM
SA018693061215byusa1114_1cytosol 0 min palmitoyl carnitine 9.6 uM
SA018694061215byusa1128_1cytosol 20 min palmitic acid 2.4 uM
SA018695061215byusa1074_1cytosol 20 min palmitic acid 2.4 uM
SA018696061215byusa1105_1cytosol 20 min palmitic acid 2.4 uM
SA018697061215byusa1129_1cytosol 20 min palmitic acid 2.4 uM
SA018698061215byusa1116_1cytosol 20 min palmitic acid 2.4 uM
SA018699061215byusa1123_1cytosol 20 min palmitic acid 2.4 uM
SA018700061215byusa1100_1cytosol 20 min palmitic acid 9.6 uM
SA018701061215byusa1080_1cytosol 20 min palmitic acid 9.6 uM
SA018702061215byusa1085_1cytosol 20 min palmitic acid 9.6 uM
SA018703061215byusa1076_1cytosol 20 min palmitic acid 9.6 uM
SA018704061215byusa1078_1cytosol 20 min palmitic acid 9.6 uM
SA018705061215byusa1093_1cytosol 20 min palmitic acid 9.6 uM
SA018706061215byusa1133_1cytosol 20 min palmitoyl carnitine 9.6 uM
SA018707061215byusa1109_1cytosol 20 min palmitoyl carnitine 9.6 uM
SA018708061215byusa1084_1cytosol 20 min palmitoyl carnitine 9.6 uM
SA018709061215byusa1068_1cytosol 20 min palmitoyl carnitine 9.6 uM
SA018710061215byusa1106_1cytosol 20 min palmitoyl carnitine 9.6 uM
SA018711061215byusa1125_1cytosol 20 min palmitoyl carnitine 9.6 uM
SA018712061215byusa1107_1mitochondria 0 min palmitic acid 2.4 uM
SA018713061215byusa1072_1mitochondria 0 min palmitic acid 2.4 uM
SA018714061215byusa1066_1mitochondria 0 min palmitic acid 2.4 uM
SA018715061215byusa1089_1mitochondria 0 min palmitic acid 2.4 uM
SA018716061215byusa1083_1mitochondria 0 min palmitic acid 2.4 uM
SA018717061215byusa1081_1mitochondria 0 min palmitic acid 2.4 uM
SA018718061215byusa1113_1mitochondria 0 min palmitic acid 9.6 uM
SA018719061215byusa1095_1mitochondria 0 min palmitic acid 9.6 uM
SA018720061215byusa1115_1mitochondria 0 min palmitic acid 9.6 uM
SA018721061215byusa1099_1mitochondria 0 min palmitic acid 9.6 uM
SA018722061215byusa1094_1mitochondria 0 min palmitic acid 9.6 uM
SA018723061215byusa1122_1mitochondria 0 min palmitoyl carnitine 9.6 uM
SA018724061215byusa1136_1mitochondria 0 min palmitoyl carnitine 9.6 uM
SA018725061215byusa1098_1mitochondria 0 min palmitoyl carnitine 9.6 uM
SA018726061215byusa1111_1mitochondria 0 min palmitoyl carnitine 9.6 uM
SA018727061215byusa1110_1mitochondria 0 min palmitoyl carnitine 9.6 uM
SA018728061215byusa1101_1mitochondria 0 min palmitoyl carnitine 9.6 uM
SA018729061215byusa1097_1mitochondria 20 min palmitic acid 2.4 uM
SA018730061215byusa1104_1mitochondria 20 min palmitic acid 2.4 uM
SA018731061215byusa1067_1mitochondria 20 min palmitic acid 2.4 uM
SA018732061215byusa1130_1mitochondria 20 min palmitic acid 2.4 uM
SA018733061215byusa1120_1mitochondria 20 min palmitic acid 2.4 uM
SA018734061215byusa1131_1mitochondria 20 min palmitic acid 9.6 uM
SA018735061215byusa1071_1mitochondria 20 min palmitic acid 9.6 uM
SA018736061215byusa1082_1mitochondria 20 min palmitic acid 9.6 uM
SA018737061215byusa1102_1mitochondria 20 min palmitic acid 9.6 uM
SA018738061215byusa1135_1mitochondria 20 min palmitic acid 9.6 uM
SA018739061215byusa1118_1mitochondria 20 min palmitoyl carnitine 9.6 uM
SA018740061215byusa1069_1mitochondria 20 min palmitoyl carnitine 9.6 uM
SA018741061215byusa1132_1mitochondria 20 min palmitoyl carnitine 9.6 uM
SA018742061215byusa1070_1mitochondria 20 min palmitoyl carnitine 9.6 uM
SA018743061215byusa1121_1mitochondria 20 min palmitoyl carnitine 9.6 uM
Showing results 1 to 68 of 68

Collection:

Collection ID:CO000408
Collection Summary:Skeletal muscle mitochondria were isolated essentially according to Chappell and Perry [Chappell JB, Perry SV (1954) Biochemical and osmotic properties of skeletal muscle mitochondria. Nature 173: 1094–1095.]. All media were ice-cold, and procedures done on ice or at 4°C. Briefly, pectoral, forelimb and hindlimb muscles were rapidly dissected and placed in basic medium [BM (mM): KCl (140), HEPES (20), MgCl2 (5), EGTA (2); pH 7.0]. Together, these muscle groups comprise a mixed population of mainly type II oxidative and glycolytic fibers. Muscle was cleaned of connective tissue and fat, minced and placed in 15 vol of homogenizing medium (HM: BM with 1 mM ATP and 1% BSA (w/v)) containing one unit of protease (Subtilisin A) per g muscle wet weight.
Collection Protocol Filename:Long-Chain_Fatty_Acid_Metabolite_Profiles_in_Skeletal_Muscle_Mitochondria.PDF
Sample Type:Mitochondria

Treatment:

Treatment ID:TR000428
Treatment Summary:Mitochondria (0.6 mg/ml) were supplied with three concentrations of palmitate corresponding to rates of β-oxidation: 1. low (2 µM) 2. medium (9 µM) 3. high (19 µM) Three ml aliquots of incubation medium [IM, (mM): KCl (120), HEPES (5), KH2PO4 (5), MgCl2 (5) and EGTA (1); pH 7.4] were supplemented with (mM) ATP (1), malate (0.05), coenzyme A (0.025), and carnitine (0.5) and added to 20-ml glass reaction vials. Solutions of low, medium and high palmitate concentrations were added to vials in a 6∶1 FA:BSA complex. Two additional incubations were performed as controls: 1. 0 µM palmitate 2. 9 µM palmitate + inhibitors
Treatment Protocol Filename:Long-Chain_Fatty_Acid_Metabolite_Profiles_in_Skeletal_Muscle_Mitochondria.PDF
Treatment Protocol Comments:The first control condition evaluated the metabolic profile of mitochondria oxidizing only malate, and included ATP, carnitine and CoA and ethanol (0.5%). The second control condition assessed effects of FA in the absence of complete oxidative catabolism, and consisted of malate, 9 µM palmitate, ATP, carnitine and CoA, and supplemented with the TCA cycle inhibitor malonate (10 mM) and the electron transport chain complex I inhibitor rotenone (5 µM)

Sample Preparation:

Sampleprep ID:SP000421
Sampleprep Summary:Tissue was homogenized using a glass/Teflon Potter-Elvehjem tissue grinder (240 rpm) and fractionated by centrifugation at 800 g (10 min), and the supernatant collected and spun at 12000 g (9 min). The pellet was resuspended in 20 ml BM and incubated on ice for 5 min (myofibrillar repolymerization). Samples were spun at 800 g (8 min) to pellet actin-myosin polymers. The supernatant was then spun at 12000 g (9 min). The final pellet was resuspended in 220 µl of BM.
Sampleprep Protocol Filename:Long-Chain_Fatty_Acid_Metabolite_Profiles_in_Skeletal_Muscle_Mitochondria.PDF
Sampleprep Protocol Comments:This isolation procedure yields mitochondria with high respiratory control ratios (state 3/state 4; ∼8–10 when supplied with 10 mM pyruvate/5 mM malate), and which are capable of activating palmitate [29], a process dependent on the integrity of enzymes on the mitochondrial outer membrane. Protein concentration was determined by a modified Lowry method with BSA as standard.
Processing Method:Homogenized

Combined analysis:

Analysis ID AN000629
Analysis type MS
Chromatography type GC
Chromatography system Leco Pegasus III GC
Column Restek Corporation Rtx-5Sil MS
MS Type EI
MS instrument type GC-TOF
MS instrument name Leco Pegasus III GC TOF
Ion Mode POSITIVE
Units counts

Chromatography:

Chromatography ID:CH000454
Instrument Name:Leco Pegasus III GC
Column Name:Restek Corporation Rtx-5Sil MS
Column Pressure:7.7 PSI
Column Temperature:50-330C
Flow Rate:1 ml/min
Injection Temperature:50 C ramped to 250 C by 12 C/s
Sample Injection:0.5 uL
Transferline Temperature:230C
Washing Buffer:Ethyl Acetate
Sample Loop Size:30 m length x 0.25 mm internal diameter
Randomization Order:Excel generated
Chromatography Type:GC

MS:

MS ID:MS000562
Analysis ID:AN000629
Instrument Name:Leco Pegasus III GC TOF
Instrument Type:GC-TOF
MS Type:EI
Ion Mode:POSITIVE
Ion Source Temperature:250 C
Ionization:Pos
Ionization Energy:70 eV
Mass Accuracy:Nominal
Source Temperature:250 C
Scan Range Moverz:85-500 Da
Scanning Cycle:17 Hz
Scanning Range:85-500 Da
Skimmer Voltage:1850 V
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